The invention relates to a stabilized antithrombin III preparation which is protected against a loss of action during pasteurization by the addition of stabilizers, and to a process for virus inactivation of an antithrombin III preparation of this type.
Antithrombin III (ATIII) is one of the most important plasmatic inhibitors. ATIII belongs to the family of serine protease inhibitors, which with their xe2x80x9ctarget proteasesxe2x80x9d enter into a complex approximating the covalent bond. This complex is very stable under physiological conditions and as a rule is rapidly eliminated from the blood circulation. The reaction between ATIII and the protease is drastically accelerated by heparin, the ATIII undergoing a slight change in conformation after association with the glycosaminoglycan and thus being able to enter into an accelerated reaction with the protease. Physiologically, these processes play a role, particularly on cell surfaces which contain glycosaminoglycans, e.g. of the heparin sulfate type, and thus represent a barrier of the cells and tissue against excessive proteolytic activity. In addition, however, plasmatic coagulation, and its regulation, is also of great importance.
The regulatory function of this inhibitor is particularly clear when the ATIII plasma levels fall, as is observed in many illnesses and particularly drastically, for example, in the case of disseminated intravascular coagulation (DIC). Even a shortfall of 70% of the corresponding plasma concentration is associated with a drastic increase in the probability of mortality. A predominance of clotting processes frequently leads to thrombotic occlusions of vessels and thus to organ failure. Correspondingly, administration of ATIII concentrates from human plasma has proven very helpful, particularly in cases of inherited and acquired deficiency states.
Patients who suffer from inherited or acquired ATIII deficiency are at present treated by substitution of concentrates obtained from human plasma. In addition to the effectiveness of these concentrates, safety with respect to the potential risk of transfer of infectious diseases must particularly be guaranteed. In addition to virus inactivation processes, such as treatment with detergents, e.g. according to the SD (solvent/detergent) method, the heat inactivation of viruses at 60xc2x0 C., which has already been used in the 1940s for albumin, is also suitable for this purpose. In general, proteins are treated at 60xc2x0 C. for up to 10 hours during pasteurization. This high temperature, however, can lead to denaturation of the proteins, which results in losses in activity and yield. In the case of ATIII, this is reflected in the loss of the heparin-binding properties of a specific protein fraction. This ATIII fraction is no longer measurable in a heparin cofactor test and is seen in two-dimensional immunoelectrophoresis as a separate peak, which can be clearly differentiated from the heparin-binding fraction.
The changes, which can mostly be attributed to conformational changes in the protein, are counteracted by addition of stabilizers to the solution to be heated. To achieve optimum protection of proteins of this type, the stabilizers employed for this must be exactly suited to each protein both in their composition and in the quantitative proportions of the individual constituents of the stabilizer mixture. Accordingly, U.S. Pat. No. 4,297,344 discloses a specific stabilizer mixture for ATIII which is used in pasteurization processes. In this patent, ATIII is mixed in aqueous solution with a carbohydrate such as sucrose and at least one amino acid from the series consisting of glycine, xcex1- and xcex2-alanine, hydroxyproline, glutamine and xcex1-, xcex2- or xcex3-butyric acid. However, completely satisfactory stabilization of ATIII cannot be thus achieved, as the heparin-nonbinding fraction is over 12% after pasteurization.
It has now been found that ATIII can be significantly more effectively protected against a loss of action during pasteurization if either one or more saccharides in a higher concentration ( greater than 1 g/ml) or one or more saccharides in combination with one or more amino acids from the group consisting of arginine, lysine, histidine, phenylalanine, tryptophan, tyrosine, aspartic acid and its salts and also glutamic acid and its salts is selected as a stabilizer. One or more of these amino acids can also be combined with glycine and/ or glutamine here.
The stabilizers are normally employed in a buffer solution, e.g. a citrate solution.
The saccharide employed can be a monosaccharide, a disaccharide or an oligosaccharide in an amount of at least 0.5 g/ml. However, a value of over 1.0 g/ml is preferred, at the same time a pH of 6.0 to 9.5, preferably of 7.0 to 8.5, being used.
The abovementioned amino acids are employed on their own or in combination in an amount of at least 0.1 mol/l, but preferably in an amount of more than 0.5 mol/l. Combinations of one or more sugars in a concentration of more than 1.5 g/ml and one or more amino acids in concentrations of more than 0.1 mol/l in each case are particularly preferred here. The combination of a sugar in an amount of more than 1.5 g/ml and one or more of the abovementioned amino acids with glycine and/or glutamine, which are all used in concentrations of over 0.2 ml in each case, is also particularly preferred. Ammonium sulfate can be added in combination with the abovementioned stabilizers up to a final concentration of 15%.
For virus inactivation, the solution of ATIII stabilized in this way is heated at 40 to 95xc2x0 C., preferably at 50 to 70xc2x0 C., for 5 to 50 hours, preferably 8 to 20 hours. However, virus inactivation at 55 to 65xc2x0 C., is most favorable.
The preparation processes for the ATIII concentrates, such as chromatographic processes by means of immobilized heparin, are known to the person skilled in the art. The process according to the invention for virus inactivation is in general applied to ATIII obtained from plasma and is dependent on the plasma fraction which is selected as a starting material for the further purification of the ATIII. The invention can likewise also be applied to recombinantly prepared or transgenic ATIII.
The stabilization described can also be used in other virus inactivation processes.